Gene transfer technology has wide-ranging utility in a number of applications relating to biological research and the treatment of disease. Central to this technology is a vector for introducing expression cassettes into target cells such that the cassettes can be expressed in the target cells. Examples of such vectors include naked DNA vectors (such as plasmids), viral vectors (such as adeno-associated viral vectors) (Berns et al., Annals of the New York Academy of Sciences, 772, 95-104 (1995)), adenoviral vectors (Bain et al., Gene Therapy, 1, S68 (1994)), herpesvirus vectors (Fink et al., Ann. Rev. Neurosci., 19, 265-87 (1996)), packaged amplicons (Federoff et al., Proc. Nat. Acad. Sci. USA, 89, 1636-40 (1992)), pappiloma virus vectors, picornavirus vectors, polyoma virus vectors, retroviral vectors, SV40 viral vectors, vaccinia virus vectors, and other vectors. Once a given type of vector is selected, its genome must be manipulated for use as a background vector, after which it must be engineered to incorporate exogenous polynucleotides.
A particularly attractive vector system employs recombinant herpes simplex virus (HSV) vectors. HSV attacks the human nervous system after a primary infection of the dermal or mucosal tissues. HSV naturally enters axonal portions of sensory ganglia and is transported to the somas of the cells, whereupon the viral DNA is released to the nucleus (Stevens, Microbiol. Rev., 53, 318-32 (1989); Roizman & Sears, in Field's Virology, 2d ed. Raven Press, Fields et al., eds. 1795-1841 (1990)). At that point, the wild-type virus either initiates a lytic cycle characterized by viral replication, or enters a latent phase. During the lytic phase, HSV gene expression proceeds through a well-characterized cascade typified by three discrete phases: Immediate Early (.alpha. or IE), Early (.beta. or E), and Late (.gamma. or L). Recombinant HSV viruses can be induced to enter latency by inactivating immediate early genes required for replication (e.g., d120, DeLuca et al., J. Virol., 56, 558 (1985)). During the latent phase, the virus persists in an episomal form for the life of its host (Mellerick and Fraser, Virology, 158, 265-75, (1987); Rock and Fraser, J. Virol., 55, 849-52, (1985)), neither interfering with neuronal function nor inducing any autoimmune response (Ramakrishnan et al., J. Virol., 68, 1864-70 (1994); Fruh et al., Nature, 375, 415 (1995)).
The HSV viral genome is well characterized, as is its life cycle, and the functions of more than 80 native coding polynucleotides are largely defined. HSV coding polynucleotides are generally contiguous linear sequences, thus facilitating genetic engineering of mutant vectors. Furthermore, as roughly half of the viral genes are dispensable for growth in cell culture, the possibility exists of deleting large segments of the HSV genome to accommodate transgenic material (Roizman & Sears, supra; Glorioso et al., in Viral Vectors, Academic Press, New York (Kaplitt & Loewy, eds.) 1-23 (1995)). Theoretically, up to 30 KB of the HSV genome can thus be replaced with exogenous material without requiring complementary host cells for propagation of the virus.
As the HSV genome is so well characterized, its genome is readily manipulated for use as a background vector. For example, it is possible to produce vectors deficient for essential loci as long as the missing translation product is otherwise provided, such as via a helper virus or complementary cell line. In fact, of the IE loci (ICP0, ICP4, ICP22, ICP27, and ICP47), only two (ICP4 and ICP27) are required for viral replication. In the absence of these loci, only other immediate early loci are expressed, and the virus is rendered replication-incompetent in non-complementing cell lines. A replication-deficient HSV virus can be induced to exist in host neural tissue in its persistent latent phase without any apparent pathological effect on the host (Ramakrishnan et al., supra), and defective HSV vectors can persist in a similar state in non-neuronal cells as well.
HSV vectors deficient for only one essential IE locus remain highly cytotoxic to infected cells, largely due to expression of the remaining IE gene products (DeLuca et al., J. Virol., 56, 558-70 (1985); Johnson et al., J. Virol., 66, 2952-65 (1992)). However, further research has indicated that mutants deleted for ICP4, ICP22, and ICP27 demonstrate reduced cytotoxicity over singly deficient vectors (see, e.g., Wu et al., J. Virol., 70(9), 6358-69 (1996)). Furthermore, the deletion of other loci reduces the cytotoxicity of HSV viruses (e.g., ICP47 (including U.sub.s 10-11), UL41, VP16, UL24; see, e.g., Jacobson et al., J. Virol., 63(4), 1839-43 (1989); Johnson, J. Virol., 68(10), 6347-62 (1994)). As such, multideficient HSV vectors represent a desirable choice for gene transfer technology.
Of course, production of multideficient viruses is one step in developing vectors for expression cassette transfer. The second step is engineering a vector containing exogenous expression cassettes. In many applications of gene transfer technology, the expression of multiple transgenes within the target cells is desirable. For example, coordinate expression of multiple cytokine sequences together with a sequence encoding an activator for an antitumor pro-drug is a potentially effective cancer therapy. Preferably, a vector is engineered such that the multiple transgenes are independently controlled (e.g., each is under regulatory control of a separate promoter) to optimize the expression kinetics. Because replication-deficient HSV vectors can transiently or constitutively express exogenous expression cassettes (Fink et al., Ann Rev. Neurosci, 19, 265-287 (1996)), HSV vectors containing multiple exogenous cassettes represent powerful agents for gene transfer applications.
Although attractive choices for gene transfer technology, HSV vectors have not been widely utilized for these therapies as of yet, primarily due to the difficulty in engineering vectors using standard procedures. The standard method for engineering mutant HSV viruses is to cotransfect host cells with the source virus and a polynucleotide comprising the desired mutation flanked by regions homologous to the target site within the HSV genome. Within the host cell, homologous recombination produces desired mutant HSV viruses less than 5% of the time, with the efficiency generally proportionate to the size of the flanking regions. Aside from inherently low efficiency, recombinant viruses often grow at markedly reduced rates vis-a-vis unmodified parental viruses within host cells, and so are easily overgrown. Thus, where the source vector is already deficient for native loci (particularly essential loci) viral growth can be substantially compromised, and the efficiency of recombination reduced accordingly. Therefore, screening plaques for desired recombinants is a laborious process, and the process becomes incrementally more tedious with multideficient HSV vectors, especially so where the desired mutation is not readily selectable.
Methods for increasing the efficiency of recombination have been attempted, but each presents significant drawbacks for quickly developing novel transfer vectors. Site-specific recombinases such as the cre-lox recombination system (reviewed in Kilby et al. Trends in Genetics, 9, 413-21 (1993)), have been employed to facilitate introduction of exogenous material into viral genomes (see, e.g., Gage et al., J. Virol., 66, 6509-15 (1992)). By these methods, exogenous polynucleotides are introduced into a recombinase recognition site previously engineered into the HSV genome. Recombinants can be selected by assaying for a reporter construct also present within the cassette. Furthermore, source viruses in which the recombinase recognition site is in a locus conferring growth benefits (e.g., the tk locus) do not enjoy the growth advantages over recombinants as seen in traditional methods. Thus, recombinase-mediated production of vectors is a more efficient method for producing HSV vectors than the co-precipitation method, routinely producing desired recombinants roughly 10% of the time, and in some instances site-specific recombination can be significantly more efficient (Rasty et al., Meth. Mol. Genet., 7, 114-30 (1995)). Despite the gain in efficiency, recombinase-mediated production of vectors presents two significant drawbacks. The first is that the method necessarily incorporates the entirety of any plasmid containing the desired insertion sequence, generally a bacterial plasmid for cloning a desired exogenous cassette. Of course, this requirement partially obviates the advantage inherent in the HSV genome's potentially large capacity to accommodate foreign DNA. Secondly, and more significantly, because the recombinase recognition site is retained (in duplicate) within a recombinant vector, subsequent rounds of site-specific recombination greatly disrupt the vectors and can result in randomization of the genome. Thus, site-specific recombination is not a preferred method for generating HSV vectors comprising multiple transgenes.
In order to enhance the efficiency of vector production, some attempts have been made to introduce unique restriction sites into the HSV genome. For example, in one system, all of the XbaI sites within the HSV genome are removed, and the RSAI site in the unique short region of the genome is changed to an XbaI site (Rixon & McLauchlan, J. Gen. Virol., 71, 2931-39 (1990)). Similarly, another system involves the introduction of a unique PacI site within the LAT region of an HSV mutant lacking 4.1 KB in one LAT copy (i.e., the HFEM mutant) (Huang et al., Gene Therapy, 1, 300-06 (1994)). With either of these systems, double-digestion of source vector DNA with the appropriate restriction enzyme permits efficient insertion of cassettes. Furthermore, screening of recombinants is achieved using a selectable marker (i.e., .beta.-galactosidase), which increases overall efficiency. However, these methods are limited to introducing exogenous DNA only at these loci, and generally only permit introduction of single cassettes.
Engineering HSV vectors to include multiple cassettes has proven difficult. Not only are the aforementioned methods inefficient for developing multigene HSV vectors, but certain properties of the HSV genome have thwarted attempts to create multiple insertions using standard means. For example, during replication, the HSV virus reacts to foreign DNA inserted at or near repeat elements. In some cases, a single expression cassette can be effectively introduced, but when the insertion of a second cassette is attempted, the virus reacts by recombining and shuffling its genome, substantially destabilizing the vector. In other instances, insertion of exogenous DNA disrupts viral function as well. Where multiple cassettes contain homologous regions to each other (i.e., a similar promoter, similar polyadenylation sequences, etc.), intragenomic recombination also can occur to destabilize the vector.
In view of the foregoing, there exists a need for a method for efficiently producing multideficient HSV vectors, especially multideficient HSV vectors that can be effectively employed in several successive rounds of mutagenesis. There also exists a need for a method for efficiently inserting exogenous polynucleotides into the HSV genome, and especially a means of creating multigene HSV vectors. There exists a further need for vectors having a multiplicity of transgenes for expression in a host cell (i.e., multigene vectors), and particularly for multigene HSV vectors having independently regulated transgenes.
The present invention provides an efficient method for creating mutant HSV vectors. The present invention further provides a multigene HSV vector having independently regulated transgenes. In particular, the present invention provides an HSV vector for cancer therapy deficient for native HSV polynucleotides and containing multiple pharmacologically active therapeutic transgenes. These and other advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.